The Xylanolytic Enzyme System of Trichoderma reesei - ACS

Chapter 46 T h e X y l a n o l y t i c E n z y m e S y s t e m o f Trichoderma

reesei 1

Kaisa Poutanen and Jurgen Puls 1

2

VTT, Biotechnical Laboratory, Tietotie 2, SF-02150 Espoo, Finland BFH, Institute of Wood Chemistry, Leuchnerstrasse 91, D-2050 Hamburg, Federal Republic of Germany

Downloaded by FUDAN UNIV on December 2, 2016 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch046

2

The xylanolytic enzyme system of Trichoderma reesei, a well-known producer of cellulolytic enzymes, is ver satile and well suited for the total hydrolysis of differ ent xylans. It consists of two major, specific and sev eral non-specific xylanases, at least one β-xylosidase, α-arabinosidase and α-glucuronidase and at least two acetyl esterases. The hydrolysis of polymeric xylans starts by the action of endoxylanases. The side-group -cleaving enzymes have their highest activities towards soluble, short xylo-oligosaccharides, and make the sub stituted oligosaccharides again accessible for xylanases and β-xylosidase. X y l a n is a n essential constituent o f h a r d w o o d s , softwoods, a n d a n n u a l p l a n t s . I n e n z y m a t i c processing o f lignocellulosic b i o m a s s , x y l a n o l y t i c e n zymes m a y be used either i n d i v i d u a l l y , i n selected m i x t u r e s for specific effects o n o n l y t h e x y l a n c o m p o n e n t o f t h e r a w m a t e r i a l , o r i n m i x t u r e s w i t h c e l l u l o l y t i c , p e c t i n o l y t i c o r a m y l o l y t i c enzymes. X y l a n s are heteropolysaccharides; accordingly, x y l a n o l y t i c enzymes i n clude different types o f endo- a n d exo-glycosidases: l , 4 - / ? - D - x y l a n a s e ( E C 3.2.1.8), /?-xylosidase ( E C 3.2.1.37), α-arabinosidase ( E C 3.2.1.55) a n d a g l u c u r o n i d a s e . I n a d d i t i o n t o these, some a c e t y l esterases are considered x y l a n o l y t i c because o f their a b i l i t y t o deacetylate x y l a n s . O f the x y l a n o l y t i c enzymes, e n d o - l , 4 - / ? - D - x y l a n a s e s have been most extensively s t u d i e d as re viewed recently (1-3). E n d o x y l a n a s e s are by d e f i n i t i o n d e p o l y m e r i z i n g e n zymes, w i t h highest a c t i v i t i e s towards l o n g c h a i n xylo-oligosaccharides o r polysaccharides. T h e h y d r o l y s i s o f the /?-l,4-linkages o f x y l a n s is c o m p l e t e d b y the a c t i o n o f /?-xylosidases, w h i c h generally have highest a c t i v i t i e s w i t h xylobiose as substrate (4-6). M u l t i p l e xylanases a n d /?-xylosidases have been observed i n different m i c r o o r g a n i s m s , e.g., o f the genera Streptomyces (7), Aspergillus (8), a n d 0097-6156/89/0399-0630$06.00/0 © 1989 American Chemical Society

Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


46.

POUTANEN & PULS

Trichoderma Xylanolytic Enzyme System

631

Trichoderma (9-10). M u c h less is k n o w n a b o u t t h e c o n c u r r e n t p r o d u c t i o n of the enzymes w h i c h cleave s u b s t i t u e n t groups o f the x y l a n p o l y m e r . T h e presence o f a c e t y l x y l a n esterases (11,12) a n d α-glucuronidases (13-15) i n x y l a n o l y t i c e n z y m e systems has o n l y recently been p o i n t e d o u t . A l t h o u g h α-arabinosidases have m a i n l y been s t u d i e d as a r a b i n a n - d e g r a d i n g enzymes (16), they have also been s h o w n t o release arabinose f r o m x y l a n s (17). W h i l e Trichoderma reesei is best k n o w n as a n efficient p r o d u c e r o f c e l l u l o l y t i c enzymes, i t has also been r e p o r t e d t o p r o d u c e x y l a n a s e a n d /?-xylosidase (18-20). T w o xylanases a n d a /?-xylosidase have been p u r i fied f r o m T. reesei (10), a n d t w o xylanases (21,22) a n d a /?-xylosidase (5) f r o m T. viride. W e have p r e v i o u s l y s h o w n t h a t T. reesei p r o d u c e s a l l t h e enzymes needed for complete h y d r o l y s i s o f n a t i v e s u b s t i t u t e d x y l a n s (23). O n e x y l a n a s e (24), a /?-xylosidase (25), a n a - a r a b i n o s i d a s e (26), a n d a n a c e t y l esterase (27) o f T. reesei have so f a r been p u r i f i e d . I n t h i s c h a p t e r , the m o d e o f a c t i o n o f these enzymes i n the h y d r o l y s i s o f different x y l a n s is discussed. Materials a n d Methods Source of Enzymes. C u l t u r e filtrates o f T. reesei s t r a i n s V T T - D - 7 9 1 2 5 a n d R u t C - 3 0 were used as s t a r t i n g m a t e r i a l for p u r i f i c a t i o n o f the i n d i v i d u a l enzymes a n d also as crude e n z y m e p r e p a r a t i o n s i n t h e h y d r o l y s i s e x p e r i m e n t s . C u l t i v a t i o n s were c a r r i e d o u t i n a l a b o r a t o r y fermentor at 3 0 ° C for 4 d o n m e d i a c o n t a i n i n g S o l k a floe cellulose ( J a m e s R i v e r C o r p . , N e w H a m p shire, U S A ) , o r glucose a n d d i s t i l l e r ' s spent g r a i n ( A l k o , L t d . , K o s k e n k o r v a , Finland). Enzyme Activity Assays. X y l a n a s e was assayed u s i n g 1% beechwood x y l a n (prepared a c c o r d i n g t o t h e m e t h o d o f E b r i n g e r o v a et ai (28)) as s u b strate as described p r e v i o u s l y (25). /?-Xylosidase was assayed u s i n g 5 m M p - n i t r o p h e n y l - / ? - D - x y l o p y r a n o s i d e as substrate (25), a n d a - a r a b i n o s i d a s e was assayed u s i n g 10 m M p - n i t r o p h e n y l - a - L - a r a b i n o f u r a n o s i d e (26). a G l u c u r o n i d a s e w a s assayed u s i n g 2 % 4 - O - m e t h y l - g l u c u r o n o s y l - x y l o b i o s e as s u b s t r a t e (13), a n d a c e t y l esterase was assayed u s i n g 1 m M a - n a p h t h y l acetate (27). Enzyme Purification. T h e p u r i f i c a t i o n o f the x y l a n o l y t i c enzymes began w i t h a d s o r p t i o n o n a c a t i o n exchanger ( C M - S e p h a r o s e F F ) at p H 4.0. T h e final p u r i f i c a t i o n w a s a c c o m p l i s h e d b y another i o n exchange step as de s c r i b e d p r e v i o u s l y for x y l a n a s e (24), /?-xylosidase (25), a - a r a b i n o s i d a s e (26) a n d a c e t y l esterase (27). Hydrolysis Experiments. T h e substrate i n the h y d r o l y s i s e x p e r i m e n t s i n c l u d e d a l k a l i - e x t r a c t e d beechwood 4 - O - m e t h y l g l u c u r o n o x y l a n , D M S O e x t r a c t e d a c e t y l a t e d beechwood 4 - O - m e t h y l g l u c u r o n o x y l a n , a l k a l i - e x t r a c t e d wheat s t r a w a r a b i n o x y l a n a n d a c e t y l a t e d or deacetylated x y l o - o l i g o m e r s f r o m s t e a m i n g o f b i r c h w o o d (24). D e a c e t y l a t i o n w a s c a r r i e d o u t b y i n c u b a t i n g the freeze-dried x y l o - o l i g o m e r s i n a m m o n i a vapor o v e r n i g h t . S u b strate c o n c e n t r a t i o n was 10 g l " , t e m p e r a t u r e 45° a n d h y d r o l y s i s t i m e 24 h. 1


632

PLANT C E L L W A L L P O L Y M E R S

Analyses. T h e m o n o - a n d disaccharides were a n a l y z e d b y H P L C (26). G e l c h r o m a t o g r a p h i c a n a l y s i s o f the x y l o - o l i g o m e r s was p e r f o r m e d i n F r a c t o g e l T S K H W - 5 0 (S) ( M e r c k , F R G ) a n d B i o g e l P 4 m i n u s 400 mesh ( B i o - R a d , U S A ) c o l u m n s (23). A c e t i c a c i d was a n a l y z e d e n z y m a t i c a l l y ( B o e h r i n g e r Test C o m b i n a t i o n 148 261).


Results a n d Discussion T h e c u l t u r e filtrates o f T. reesei contained a large n u m b e r o f b o t h c e l l u l o l y t i c a n d h e m i c e l l u l o l y t i c enzymes, w h i c h c o u l d be p a r t i a l l y separated b y c h r o m a t o f o c u s s i n g ( F i g . 1). O f t h e c e l l u l o l y t i c enzymes, several e n d o g l u canases a n d t w o cellobiohydrolases have a l r e a d y been i s o l a t e d a n d c h a r a c terized (30). S o m e o f the endoglucanases i s o l a t e d are nonspecific a n d have x y l a n a s e a c t i v i t y (31). T h e t w o m a j o r x y l a n a s e ( X y l ) peaks i n F i g u r e 1 corresponded t o p l - v a l u e s o f above 7.5 a n d 5.5. W h e n t h e former e n z y m e was f u r t h e r p u r i f i e d (24), i t s isoelectric p o i n t was f o u n d t o b e a b o u t p i 9 ( T a b l e I ) . P r e v i o u s l y , the presence o f at least three e l e c t r o p h o r e t i c a l l y dif ferent xylanases i n T. reesei c u l t u r e filtrates w a s r e p o r t e d , w i t h t h e m o s t a c i d i c one s h o w n t o have b r o a d substrate specificity (10). T h i s is i n agree m e n t w i t h the r e p o r t e d occurrence o f three xylanases ( p i > 7, p i 5.1, p i 4.5) i n T. reesei (32). T h e results o f t h i s s t u d y , together w i t h those o f others (10,20,31-33) suggest t h a t t h e enzyme s y s t e m o f T. reesei c o n t a i n s two m a j o r , specific endoxylanases, w i t h isoelectric p o i n t s o f 8.5-9 a n d 5-5.5, a n d m a n y non-specific endoglycanases w i t h m o r e a c i d i c p l - v a l u e s a n d b o t h 1,4-/?-glucanase a n d l,4-/?-xylanase a c t i v i t i e s . T a b l e I. X y l a n o l y t i c E n z y m e s Isolated f r o m Trichoderma

Enzyme Xylanase /?-Xylosidase a-Arabinosidase A c e t y l esterase a

b

reesei

M W (kDa)

Pi*

pH-Optimum

Reference

20 100 53 45

~9 4.7 7.5 6.8

5.3 4.0 4.0 5.5

24 25 26 27

From S D S - P A G E . F r o m chromatofocussing.

T h e /?-xylosidase o f T. reesei was a r a t h e r large, p r o b a b l y d i m e r i c e n z y m e a n d , like most other /?-xylosidases, h a d a n acidic p i - v a l u e ( T a b l e I ) . A /?-xylosidase purified earlier f r o m T. viride w i t h a m o l e c u l a r weight o f 101 k D a also h a d a n isoelectric p o i n t p i 4.45 (5). I n a d d i t i o n t o p - n i t r o p h e n y l /?-xylopyranoside a n d xylo-oligosaccharides, t h e /?-xylosidase o f T. reesei also showed a c t i v i t y w i t h p - n i t r o p h e n y l - a - a r a b i n o f u r a n o s i d e as s u b s t r a t e , b u t d i d n o t h y d r o l y z e a r a b i n a n o r release arabinose f r o m a r a b i n o x y l a n . T h e purified α-arabinosidase, however, was capable o f effecting b o t h the l a t t e r hydrolyses (26, T a b l e II). T h e isoelectric p o i n t o f the α-arabinosidase o f T. reesei w a s p i 7.5 a n d i t s m o l e c u l a r weight 53 k D a ( T a b l e I ) .


46.

POUTANEN & PULS



CBH II

633

Xyl, EQ

Xyl, Ara, BQ pH 8

salt elutlon

Elution volume

Figure 1. Fractionation of proteins in the culture filtrate of Trichoderma reesei according to their pi values: Xyl, xylanase; Ara, arabinosidase; AE, acetyl esterase; βΧ, /?-xylosidase; aG, α-glucuronidase; fiG, /?-glucosidase; CBH, cellobiohydrolase; EG, endoglucanase. Chromatofocusing was performed in a PBE-94 anion exchange resin (Pharmacia) with a pH-gradient created by ampholyte buffers (Pharmacia). Solid line, A ^ ; dotted line, pH. (Reproduced with permission from ref. 24. Copyright 1988.)


634

PLANT C E L L W A L L POLYMERS

T a b l e I I . T h e effect o f α-arabinosidase o n the h y d r o l y s i s o f wheat s t r a w a r a b i n o x y l a n . S u b s t r a t e c o n c e n t r a t i o n 10 g l " , i n i t i a l p H 4, t e m p e r a t u r e 45° C , h y d r o l y s i s t i m e 24 h 1

Hydrolysis Products (% of substrate)

Enzyme Activities ( n k a t / g substrate)


Xylanase 25000 25000 25000 25000 25000* a

b

0

/?-Xylosidase 0 160 160 160 160*

e

a - Arabinosidase"

Xylose

Arabinose

0 0 1400 14000 1300*

4 37 43 56 56

0 0 4 7 7

P u r i f i e d enzymes o f T. reesei. A c t i v i t i e s o f crude T. reesei c u l t u r e filtrate.

T h e h y d r o l y s i s p r o d u c t s o f three different x y l a n s b y t h e 20 k D a x y lanase o f T. reesei varied a c c o r d i n g t o t h e n a t u r e a n d degree o f s u b s t i t u t i o n of the substrate ( F i g . 2a-c). T h e role o f a c e t y l groups i n t h e accessibility o f beechwood x y l a n was evident ( F i g . 2 a a n d 2 b ) : T h e a c e t y l a t e d s u b s t r a t e was m o r e soluble i n water, more u n i f o r m l y a t t a c k e d b y t h e e n z y m e a n d y i e l d e d a w i d e r range o f h y d r o l y s i s p r o d u c t s ( F i g . 2 a ) . O n t h e other h a n d , the h y d r o l y z a t e o f the n o n - a c e t y l a t e d 4 - O - m e t h y l - g l u c u r o n o s y l - s u b s t i t u t e d beech x y l a n contained some insoluble residue (not v i s i b l e i n the c h r o m a t o g r a m ) a n d large soluble oligomers, w h i c h were eluted i n the v o i d v o l u m e ( X ) ( F i g . 2 b ) . X y l o b i o s e a n d s u b s t i t u t e d oligomers were the m a i n s o l uble h y d r o l y s i s p r o d u c t s . T h e presence o f 4 - 0 - m e t h y l g l u c u r o n o - s u b s t i t u t e d xylo-oligosaccharides was verified b y a n i o n exchange c h r o m a t o g r a p h y (14) (results n o t s h o w n ) . T h e h y d r o l y z a t e o f a r a b i n o x y l a n resembled t h a t o f g l u c u r o n o x y l a n ( F i g . 2c). T h e a c c u m u l a t i o n o f 4 - O - m e t h y l - g l u c u r o n o s y l - a n d a r a b i n o s y l s u b s t i t u t e d xylo-oligosaccharides has also been reported w h e n u s i n g x y l a n a s e s f r o m other sources (34,35). T h e role o f a r a b i n o s y l substituents a n d the need for α-arabinosidase i n the p r o d u c t i o n o f xylose f r o m a r a b i n o x y l a n was also deduced f r o m the results i n T a b l e I I . W h e n the purified 20 k D a x y l a n a s e a n d /?-xylosidase were used i n the h y d r o l y s i s , the xylose y i e l d was o n l y 6 6 % o f t h a t p r o d u c e d by the whole c u l t u r e filtrate at the same a c t i v i t y levels, a n d no arabinose was p r o d u c e d . A d d i t i o n o f α-arabinosidase increased the yields o f b o t h xylose a n d arabinose. n

T h e h y d r o l y s i s o f acetylated x y l o - o l i g o m e r s f r o m the s t e a m i n g e x t r a c t of b i r c h w o o d u s i n g a n enzyme m i x t u r e c o n t a i n i n g o n l y x y l a n a s e a n d βx y l o s i d a s e was very l i m i t e d ( T a b l e I I I ) . C h e m i c a l d e a c e t y l a t i o n showed t h a t the substrate specificities o f x y l a n a s e a n d /?-xylosidase w i t h respect to substrate D P overlapped, a n d t h a t /3-xylosidase also h y d r o l y z e d longer oligosaccharides t h a n xylobiose t o xylose. T h e a d d i t i o n o f a c e t y l esterase to the h y d r o l y s i s o f a c e t y l a t e d x y l o - o l i g o m e r s enhanced x y l o s e p r o d u c t i o n ( T a b l e I I I ) , b u t d e a c e t y l a t i o n was s t i l l i n c o m p l e t e (the a c e t y l content o f


46.

POUTANEN & PULS


635

c φ c ο υ Φ Φ

>> .C Ο 13 k.

φ

ϋ

υ


Elution volume *2

MGA

c φ c ο υ Φ Φ

•σ >»

ο

-Ω L_

Φ

Ο

Elution volume c Φ c ο ο φ φ

k.

"Ο

>s .c ο φ

ϋ

Elution volume c F i g u r e 2. H y d r o l y s i s p r o d u c t s o f beechwood O - a c e t y l g l u c u r o n o x y l a n ( F i g . 2a), beechwood g l u c u r o n o x y l a n ( F i g . 2 b ) , a n d wheat s t r a w a r a b i n o x y l a n ( F i g . 2c), as a n a l y z e d b y gel p e r m e a t i o n c h r o m a t o g r a p h y . T h e h y d r o l y s i s was c a r r i e d o u t at p H 5 at 4 5 ° C for 24 h u s i n g 10.000 n k a t o f the 20 k D a x y l a n a s e o f T. reesei. X = xylose; X = x y l o b i o s e ; XMGA = 4-O-methylg l u c u r o n o s y l s u b s t i t u t e d xylo-oligosaccharides; X = x y l o - o l i g o s a c c h a r i d e s D P > 20. 2

n


636

PLANT CELL WALL POLYMERS

substrate A was 1 0 % o f the d r y weight). T h i s p h e n o m e n o n is b e i n g f u r t h e r s t u d i e d . It is possible t h a t t h e p u r i f i e d esterase specifically removed acetic a c i d f r o m o n l y one p o s i t i o n o n the x y l o p y r a n o s e r i n g ( u n p u b l i s h e d results). T a b l e I I I . H y d r o l y s i s o f a c e t y l a t e d ( A ) a n d c h e m i c a l l y deacetylated ( B ) x y l o - o l i g o m e r s i n t h e s t e a m i n g e x t r a c t o f b i r c h w o o d b y p u r i f i e d enzymes o f T. reesei. S u b s t r a t e c o n c e n t r a t i o n 10 g l ~ \ i n i t i a l p H 5, t e m p e r a t u r e 4 5 ° C , h y d r o l y s i s t i m e 24 h Enzyme Activities (nkat g " )


1

Substrate

Xylanase

/?-Xylosidase

Acetyl Esterase

A Β

5000 5000

— —

A Β

— —

500 500

— — — —

A A Β

5000 5000 5000

500 500 500

— 500

—

Hydrolysis Products (% o f d r y weight)

Xylose

Xylo biose

Acetic Acid

2 3

5 25

0.7

7 21

< 1 < 1

0.7

9 18 33

0 < 1 0

0.9 3.3

— —

—

S y n e r g i s m between the different x y l a n o l y t i c enzymes was f u r t h e r d e m o n s t r a t e d w h e n a p a r t i a l l y purified e n z y m e p r e p a r a t i o n was used as a source o f xylanase a c t i v i t y ( T a b l e I V ) . T h i s p r e p a r a t i o n , s e p a r a t e d f r o m T. reesei c u l t u r e f i l t r a t e b y gel c h r o m a t o g r a p h y (10), c o n t a i n e d b o t h t h e 20 k D a x y l a n a s e , another glycanase w i t h h i g h l a m i n a r i n a s e a n d low x y l a n a s e a c t i v i t y , a n d a h i t h e r t o u n i d e n t i f i e d esterase. W h e n s u p p l e m e n t e d w i t h /?-xylosidase, the xylanases a n d esterase o f t h i s p r e p a r a t i o n released a b o u t h a l f o f t h e xylose a n d acetic a c i d o f the substrate ( T a b l e I V ) . W h e n p u r i fied a c e t y l esterase was a d d e d , the yields o f xylose a n d acetic a c i d increased f u r t h e r a b o u t 1.6-fold. Because the α-glucuronidase o f T. reesei has n o t y e t been p u r i f i e d , a c u l t u r e filtrate o f Agaricus bisporus, w i t h h i g h α-glucuronidase a c t i v i t y (14) w a s used t o complete the s y n e r g i s m . T h e a d d i t i o n o f α-glucuronidase cleaved off 4 - O - m e t h y l g l u c u r o n i c a c i d a n d m a d e the oligomers accessible for /?-xylosidase a n d esterases. U s i n g t h i s m i x t u r e , t h e x y l o s e y i e l d w a s increased t o the same value as t h a t o b t a i n e d w i t h the whole c u l t u r e filtrate. Conclusions T h e x y l a n o l y t i c e n z y m e s y s t e m o f T. reesei consists o f several e n d o x y lanases, at least three different exoglycosidases a n d at least t w o a c e t y l es terases. These enzymes c o n t r i b u t e t o the h y d r o l y s i s o f p l a n t cell w a l l s i n m a n y o f the a p p l i c a t i o n s o f c e l l u l o l y t i c T. reesei e n z y m e p r e p a r a t i o n s . A s c h e m a t i c figure o f t h e suggested h y d r o l y s i s m e c h a n i s m o f x y l a n s b y the


46.

POUTANEN & PULS


637

T a b l e I V . E n z y m a t i c H y d r o l y s i s o f the H i g h - M o l e c u l a r F r a c t i o n o f S t e a m e d B i r c h w o o d X y l a n . T h e substrate w a s f r a c t i o n a t e d b y u l t r a f i l t r a t i o n p r i o r t o h y d r o l y s i s t o remove i m p u r i t i e s a n d t h e 1-5 D P oligosaccharides. S u b s t r a t e c o n c e n t r a t i o n 10 g l " , i n i t i a l p H 5, t e m p e r a t u r e 4 5 ° C , h y d r o l y s i s t i m e 24 h 1

Enzyme Activities ( n k a t / g substrate)


Xylanase" 12000 0 0 0 12000 12000 12000 15000 " e

α

6

c

d

Hydrolysis Products (% o f substrate)

β-Xylosidase*

Acetyl Esterase*

α-Glucuro nidase

Xylose

Acetic Acid

0 500 0 0 500 500 500 500

0 0 500 0 0 500 500 500

0 0 0 30 0 0 30 20

6 6 0 1 25 42 53 53

4.5 0.5 1.2 0.5 5.9 9.4 11.5 12.1

a

a

c

c

a

P a r t l y purified p r e p a r a t i o n f r a c t i o n a t e d f r o m T. reesei c u l t u r e b y gel c h r o m a t o g r a p h y (10). P u r e enzymes o f T. reesei. Agaricus bisporus c u l t u r e filtrate. T. reesei c u l t u r e filtrate.

filtrate

enzymes o f T. reesei is s h o w n i n F i g u r e 3. T h e h y d r o l y s i s starts b y t h e a c t i o n o f endoxylanases, w h i c h decreases t h e average D P o f t h e s u b s t r a t e . T h e s i d e - g r o u p - c l e a v i n g enzymes have t h e i r highest a c t i v i t y t o w a r d s s o l u b l e , short xylo-oligosaccharides, a n d t h e h y d r o l y s i s is c o m p l e t e d b y t h e synergistic a c t i o n o f b o t h side-group a n d b a c k b o n e - c l e a v i n g enzymes. In a d d i t i o n t o c e l l u l o l y t i c a n d x y l a n o l y t i c enzymes t h e hydrolases p r o duced b y T. reesei have also been r e p o r t e d t o i n c l u d e m a n n a n a s e , p e c t i nase, amyloglucosidase a n d protease (36,37). T h e present a n d p o t e n t i a l a p p l i c a t i o n s o f T. reesei enzymes i n c l u d e t o t a l h y d r o l y s i s o f l i g n o c e l l u l o s i c m a t e r i a l s t o glucose a n d / o r x y l o s e , s t i m u l a t i o n o f g e r m i n a t i o n i n m a l t i n g , m o r e c o m p l e t e s a c c h a r i f i c a t i o n o f cereals i n g r a i n a l c o h o l p r o d u c t i o n a n d i m p r o v e m e n t o f t h e storage properties a n d d i g e s t i b i l i t y o f silage feed. I n these a p p l i c a t i o n s s y n e r g i s m is needed n o t o n l y between t h e i n d i v i d u a l x y l a n o l y t i c or c e l l u l o l y t i c enzymes t o h y d r o l y z e x y l a n o r cellulose, b u t p r o b a b l y also between different e n z y m e groups for successive h y d r o l y s i s o f the p l a n t cell w a l l m a t r i x . D u e t o i t s v e r s a t i l i t y , T. reesei is a n excellent source o f enzymes i n these cases. I n a p p l i c a t i o n s where selective h y d r o l y s i s is r e q u i r e d , however, the e n z y m e s p e c t r u m s h o u l d be t a i l o r e d for a p a r t i c u l a r purpose. T h i s c o u l d be achieved b y a d j u s t i n g e n z y m e p r o d u c t i o n c o n d i t i o n s , b y f r a c t i o n a t i n g t h e enzymes or b y m o l e c u l a r c l o n i n g .



638

PLANT C E L L W A L L P O L Y M E R S

O-Acetyl-Glucuronoxylans Arabinoxylans Arabinoglucuronoxylans

\r

Endo-e-1,4-xylanases

Different soluble (substituted) xylo-oligosaccharides Endo-e-1,4-xylanases £-D-xylosidase a-L-arabinosidase a-D-glucuronidase ^ Acetyl esterases Xylose Arabinose 4-o-methylglucuronic acid Acetic acid

F i g u r e 3. T e n t a t i v e h y d r o l y s i s m e c h a n i s m o f different x y l a n s b y the x y l a n o l y t i c enzymes o f T. reesei.


46.

POUTANEN & PULS


639


Literature Cited 1. Woodward, J . Top. Enz. Ferment.Biotechnol.1984, 8, 9-30. 2. Dekker, R. F. H. In Biosynthesis and Biodegradation of Wood Compo nents; Higuchi, T . , Ed.; Academic Press: Orlando, FL, 1985; p. 505. 3. Wong, K. K. Y.; Tan, L . U. L.; Saddler, J . N. Microbiol. Rev. 1988, 52, 305-17. 4. Rodionova, Ν. Α.; Tavobilov, I. M . ; Bezborodov, A . M . J. Appl. Biochem. 1983, 5, 300-12. 5. Matsuo, M.; Yasui, T . Agric. Biol. Chem. 1984, 48, 1845-60. 6. Matsuo, M . ; Fujie, Α.; Win, M.; Yasui, T . Agric. Biol. Chem. 1987, 51, 2367-79. 7. Marui, M.; Nakanishi, K.; Yasui, T . Agric. Biol. Chem. 1985, 49, 33993407. 8. Fournier, R.; Frederick, M. M.; Frederick, J . R.; Reilly, P. J . Biotechnol. Bioeng. 1985, 27, 539-46. 9. Wong, Κ. K. Y.; Tan, L . U. L.; Saddler, J . N. Enz. Microb. Technol. 1986, 8, 617-22. 10. Lappalainen, A. Biotechnol. Appl. Biochem. 1986, 8, 437-48. 11. Biely, P.; Puls, J.; Schneider, H. FEBS Lett. 1985, 186, 80-84. 12. Biely, P.; MacKenzie, C . R.; Puls, J . ; Schneider, H. Bio/Technology 1986, 4, 731-33. 13. Puls, J . ; Poutanen, K.; Schmidt, O.; Linko, M . Proc. 3rd Intl. Conf. Biotechnol. in the Pulp and Paper Industry; 1986, p. 93. 14. Puls, J.; Schmidt, O.; Granzow, C. Enz. Microb. Technol. 1987, 9, 83-88. 15. Ishihara, M.; Shimizu, K. Mokuzai Gakkaishi 1988, 34, 58-64. 16. Kaji, A. Adv. Carbohydr. Chem. Biochem. 1984, 42, 383-94. 17. Andrewartha, Κ. Α.; Phillips, D. R.; Stone, B. A. Carbohydr. Res. 1979, 77, 191-204. 18. Tangnu, S. K.; Blanch, H. W.; Wilke, C. R. Biotechnol. Bioeng. 1981, 23, 1837-49. 19. Dekker, R. F. H. Biotechnol. Bioeng. 1983, 25, 1127-46. 20. Hrmova, M.; Biely, P.; Vrsanska, M . Arch. Microbiol. 1986, 144, 30711. 21. Sinner, M.; Dietrichs, Η. H. Holzforschung 1975, 29, 207-14. 22. Gibson, T . S.; McCleary, Β. V . Carbohydr. Polym. 1987, 7, 225-40. 23. Poutanen, K.; Rättö, M.; Puls, J.; Viikari, L. J. Biotechnol. 1987, 6, 49-60. 24. Poutanen, K. Ph.D. Thesis, Technical Research Centre of Finland, Publications 47, Espoo, 1988. 25. Poutanen, K.; Puls, J . Appl. Microbiol. Biotechnol. 1988, 28, 425-32. 26. Poutanen, K. J. Biotechnol. 1988, 7, 271-82. 27. Poutanen, K.; Sundberg, M . Appl. Microbiol. Biotechnol. 1988, 28, 419-24. 28. Ebringerova, Α.; Kramav, Α.; Rendos, F.; Domansky, R. Holzforschung 1967, 21, 74-77. 29. Puls, J . ; Poutanen, K. Körner, H.-U.; Viikari, L. Appl. Microbiol. Biotechnol. 1985, 22, 416-23.


640

PLANT CELL WALL POLYMERS


30. Teeri, T . Ph.D. Thesis, Technical Research Centre of Finland, Publi cations 38, Espoo, 1987. 31. Biely, P.; Markovic, O. Biotechnol. Appl. Biochem. 1988, 10, 99-106. 32. Esterbauer, H.; Hayn, M.; Tuisel, H.; Mahnert, W. Holzforschung 1983, 37, 601-08. 33. Kolarova, N.; Farkas, V. Biologia (Bratislava) 1983, 38, 721-25. 34. Sinner, M.; Dietrichs, H. H. Holzforschung 1976, 30, 50-59. 35. Kusakabe, I.; Ohgushi, S.; Yasui, T.; Kobayashi, T . Agric.Biol.Chem. 1983, 47, 2713-23. 36. Bailey, M . J.; Nevalainen, Κ. M . H. Enzyme Microb. Technol. 1981, 3, 153-57. 37. Haltmeier, T . ; Leisola, M . ; Ulmer, D.; Waldner, R.; Fiechter, A. Biotechnol. Bioeng. 1983, 25, 1685-90. RECEIVED May 19, 1989


The Xylanolytic Enzyme System of Trichoderma reesei - ACS

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